Alcoholism is a major health and economic problem which imposes broad reaching concerns not only to the afflicted individuals, but to society at large. In the United States alone, at least ten to twenty million people are classified as chronic alcoholics and long-term alcohol abusers. In addition, other countries have serious problems with chronic alcohol consumption as well as with the objective diagnosis and detection of long-term alcohol use.
For example, in the United States, health and medical costs related to alcohol abuse are continually increasing and are estimated to be on the order of 31.6 billion dollars annually. In 1980, lost employment and reduced productivity due to costs related to alcohol abuse were estimated to be approximately 54.7 billion dollars. In 1983, this figure jumped to 116.9 billion dollars, 60% of which was attributed to lost employment and reduced productivity, and 13% of which was attributed to health care costs. Furthermore, at least 1.5 million Americans participate in alcohol rehabilitation programs and another 0.5 million chronic alcoholics are in contact with medical and treatment professionals. Thus, at least 2 million people, at a minimum, could conceivably use the test methods of the present invention several times during the course of rehabilitation.
It is known that chronic (i.e. over a period of weeks, months, or longer) alcoholic and long-term alcohol users rarely admit their excessive consumption of alcohol. In spite of attempts to standardize the diagnosis of alcohol abuse and chronic alcoholism based on operational and functional criteria, many problems exist in the detection and diagnosis of alcohol-related disorders. One major problem is that patient cooperation is required, and often, alcoholics do not approach their physicians to ask for help specifically to stop their excessive or pathological drinking. Unfortunately, even when questioned directly by their physicians, alcoholics rarely disclose the true extent of their alcohol consumption, and often deny and minimize any association between their use of alcohol and their other symptoms or problems. Because it is difficult to detect and diagnose alcoholism and alcohol abuse in patients, physicians frequently misdiagnose or under-diagnose alcohol-related disorders.
As evidenced by the foregoing explanation, there is a real need for simple and reliable tests which could help physicians in their desire and ability to detect alcoholism and monitor alcohol abuse. An early diagnosis of chronic alcohol abuse would also allow constructive intervention therapy at a stage when predicted recovery from alcoholism would be more favorable. Moreover, improvements in the ability to monitor the extent of abstinence in treated alcoholics would permit researchers to better assess the efficacy of various treatment regimens for chronic alcoholism.
Thus, biological and/or biochemical markers which serve as objective, physiological measures of alcohol consumption are extremely important for patient clinical care and research when a valid drinking history is required. In addition, reliable, reproducible, and economical methods of detecting and monitoring these biomarkers are needed. Such biological markers and detection methods would provide treatment professionals and researchers with an unbiased and credible tool for monitoring patient compliance with treatment goals. In addition, there is a need for objective and dependable biomarkers of alcohol consumption to aid in the diagnosis and treatment of conditions other than alcoholism when alcohol consumption is suspected.
Until the present invention, a simple, reliable, and objective biomarker of chronic alcohol consumption and the methods of detecting and monitoring the presence of such a biomarker in test individuals have been rather limited in scope and have frequently lacked sensitivity and ease of detection and use. An ideal biomarker for chronic alcohol consumption would be a direct derivative of alcohol that responds to and appears only after continued chronic intake of alcohol. Until the present invention, such a useful biomarker, which is a direct product of heavy alcohol consumption, had not been discovered or previously described to be directly correlated with chronic alcohol consumption, or to be readily detected over prolonged time periods following an individual's last drink.
Many current biological indicators of alcohol consumption are severely hampered by the fact that alcohol is fully and quickly eliminated from the body through oxidative metabolism. Thus, the measurement of alcohol in and of itself as a biomarker either in breath or in body fluids indicates only the very recent consumption of alcohol. Because of the rapid rate of oxidative metabolism, the direct detection of alcohol per se in breath or body fluids can be assessed only within a few hours of a drinking episode. Thus, the direct measurement of alcohol in a subject will often fail to detect drinking or alcohol consumption, even in an individual who has consumed alcohol over an extended period of time, but who has abstained during the most recent 24 hour period.
Unlike the present invention, a number of enzyme and blood chemistry assays, which are available in the clinical laboratory and have been used to detect heavy alcohol consumption, are not readily adaptable for routine, accurate, rapid, and cost-effective diagnostic testing. Previously reported assays also suffer from loss or degradation of the product being monitored over a short time period. Representative examples include gamma-glutamyltranspeptidase (GGT) (Rosalki, S. B. and Rau, D. (1972). Clinica Chemica Acta, 39:41; Rollason, J. G. et al. (1972). Clinica Chemica Acta, 39:75); aspartate aminotransferase (AST) (Bernadt, M. W., (1982). Lancet, 1:325); alanine aminotransferase (ALT); glutamate dehydrogenase (GDH) (Schellenberg, F., (1983). Ann. Biol. Clin., 41:255); mean corpuscular volume (MCV) (Whitfield et al. (1978). Ann. Clin. Biochem.: 15, 297-303); and alpha-amino-n-butyric acid/leucine ratio (AANB/L). Several of these assays are sometimes combined to increase the discriminating power of each test separately; however, the applicability of these biochemical parameters to routine clinical use is limited by the lack of overall specificity, sensitivity, and reliability of the tests. GGT, one of the most commonly relied upon biochemical measures of heavy alcohol use, is elevated in patients with liver disease of both non-alcoholic and alcoholic origin. Furthermore, increased levels of GGT can be found not only as a consequence of obesity, pancreatic disease, diabetes, hyperlipidemia, heart disease, smoking and trauma, but also as a consequence of the use of drugs which induce microsomal enzymes, particularly anti-epileptic and anti-coagulant drugs. The AANB/L ratio also appears to be influenced by alcoholic and non-alcoholic liver disease. Moreover, the requirement of an amino acid analyzer to perform the assays makes many of these tests expensive and less conducive to routine screening and diagnostic procedures.
Other methods for the biochemical diagnosis of chronic alcoholism suffer from practical limitations and inefficient features. For example, urinary dolichol levels have been suggested to be a biomarker for chronic alcoholism (Pullarkat, R. K. and Raguthu, S., (1985). Alcohol. Clin. Exp. Res. 9: 229-231). This finding has been confirmed by other laboratories (Roine, R. P. et al., (1987). Alcohol. Clin. Exp. Res. 11: 525-527; Salaspuro, M. P., et al., (1987). In: Genetics and Alcoholism, Ed. Alan R. Liss, Inc., 231-240; Roine, R. P., (1988). Alcohol 5: 229-231). Estimation of the extremely small levels of urinary dolichol that are able to be detected requires the use of sophisticated equipment such as high performance liquid chromatography (HPLC). Thus, dolichol is not easily adaptable for rapid and facile clinical evaluation and testing. Furthermore, the appearance of dolichol, like other known markers of alcohol consumption, results from secondary effects of alcohol on metabolism. Since dolichol is present only in small amounts (e.g. nanograms/milliliter) in the urine of chronic alcoholics, it is difficult to detect and assess accurately without using large amounts of starting sample, elaborate sample preparation techniques, and expensive clinical research instruments.
Measurement of acetaldehyde-protein adducts in urine or other body fluids by HPLC or in body fluids and cells by an immunoassay method has been reported as methods for detecting chronic alcoholism (Tang, B. K. et al., (1986). Biochem. Biophys. Res. Comm. 140: 924-927; Israel, Y. et al., (1986). Proc. Natl. Acad. Sci. 83: 7923-7927; U.S. Pat. No. 4,900,664 to Y. Israel and R. Arnon, filed Oct. 2, 1986). However, although these adducts are not secondary byproducts of alcohol, they are known to be very unstable and to decompose completely after 24 hours. The HPLC method also requires the use of sophisticated equipment which is cumbersome and not easily adaptable for routine, clinical testing.
The reduction of monoamine oxidase in platelets of chronic alcoholics has been demonstrated (Tabakoff et al., (1988). New Engl. J. Med. 318: 134-149). However, the changes observed are marginal and the detection procedures are very time-consuming to perform, since platelet membranes must be prepared and various enzyme assays must be conducted.
Carbohydrate deficient-species of transferrin in serum has been demonstrated to be a specific marker for chronic alcohol consumption (Stibler, H. et al., (1986). Alcohol. Clin. Exp. Res. 10 (5):535-544; Behrens, U. J. et al., (1988). Alcohol. Clin. Exp. Res. 12 (3):427-432; Storey, E. L. et al., (1987). Lancet 1: 1292). The methods used involve an isocratic anion exchange chromatography of isotransferrins followed by a double antibody transferrin radioimmune assay. These procedures are quite laborious to perform and cannot always be readily adapted as rapid clinical tests.
Commonly-occurring sugars in biological fluids have been separated and quantified by a one-dimensional thin layer chromatography (TLC) method (Zilik, Z. et al., (1979). J. Chromatog. 164:91-94). This method uses a reagent comprised of diphenylamine, aniline, and phosphoric acid to detect the normally low levels of known sugars commonly-found in human urine and plasma, as well as to detect glucose present in the urine of diabetics. Zilic et al. do not contemplate either the detection of chronic alcoholism or the presence of a novel biomarker linked directly to chronic alcohol consumption.
Ethyl glucuronides have been reported following the metabolism of alcohol in animals and man (Kamil, I. A. et al., (1952). Biochem. J. 51:32-33; P. I. Jaakonmaki et al., (1967). Eur. J. Clin. Pharmacol. 1:67-70; T. Kozu, (1973), Shinsu Medical J. 6:595-601; Schmitt, G. et al., (1995). J. Analyt. Toxicol. 19:91-94). However, such reports do not provide reliable and safe methods of detecting chronic alcohol consumption and do not recognize that ethyl-.beta.-glucuronide serves as an effective biomarker of alcoholism and is detected in an individual's body fluid sample from about 24 hours to over 14 days after that individual has taken a last drink.